7570 J. Med. Chem. 2009, 52, 7570–7579 DOI: 10.1021/jm9004225
Probes for Narcotic Receptor Mediated Phenomena. 39.1 Enantiomeric N-Substituted Benzofuro[2,3-c]pyridin-6-ols: Synthesis and Topological Relationship to Oxide-Bridged Phenylmorphans2,† Yi Zhang,‡ Yong Sok Lee,¥ Richard B. Rothman,z Christina M. Dersch,z Jeffrey R. Deschamps,§ Arthur E. Jacobson,‡ and Kenner C. Rice*,‡ ‡
Drug Design and Synthesis Section, Chemical Biology Research Branch, National Institute on Drug Abuse and the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Department of Health and Human Services, 5625 Fishers Lane, Room 4N03, Bethesda, Maryland 20892-9415, zClinical Psychopharmacology Section, Chemical Biology Research Branch, National Institute on Drug Abuse, Addiction Research Center, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland 21224, §Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375, and ¥Center for Molecular Modeling, Division of Computational Bioscience, CIT, National Institutes of Health, DHHS, Bethesda, Maryland 20892 Received April 1, 2009
Enantiomers of N-substituted benzofuro[2,3-c]pyridin-6-ols have been synthesized, and the subnanomolar affinity and potent agonist activity of the known racemic N-phenethyl substituted benzofuro[2,3c]pyridin-6-ol can now be ascribed to the 4aS,9aR enantiomer. The energy-minimized structures suggest that the active enantiomer bears a greater three-dimensional resemblance to morphine than to an ostensibly structurally similar oxide-bridged phenylmorphan. Structural features of the conformers of N-substituted benzofuro[2,3-c]pyridin-6-ols were compared to provide the rationale for their binding affinity. Introduction 3
In 1989, Hutchison, et al. published the synthesis of racemic N-substituted benzofuro[2,3-c]pyridin-6-ols and noted that one of them, the N-phenethyl derivative of the cis-benzofuropyridin-6-ol had high μ-opioid affinity (Ki = 0.9 nM) and potent antinociceptive activity. These compounds appeared to have marked structural resemblance to the oxidebridged phenylmorphans. We published our initial work on the synthesis of the 12 racemic topologically rigid N-methylsubstituted a-through-f-oxide-bridged phenylmorphans (Figure 1) in 19844,5 and have recently reported the preparation of the final pair of oxide-bridged phenylmorphans.4-13 Some of the N-phenethyl analogues have been synthesized and their pharmacological activity determined.12,13 A few of the enantiomers11 of the racemates have also been examined. The topology of the ortho-b, -d,a, and -f isomers has been reported,13 and comparisons have previously been made to a highly potent azabicyclo[3.3.1]nonane μ-opioid agonist.14 We believed that we could gain information about the threedimensional shape of a molecule required for it to interact with a specific opioid receptor if any of the 12 oxide-bridged racemic phenylmorphans was found to have agonist or antagonist activity. There was no way then, or now, to determine the agonist or antagonist conformation of a ligand acting at an opioid receptor.
In order to gain additional insight into the ligand-receptor interaction, we thought it would prove useful to find new rigid ligands with relatively few conformational shapes that could interact with opioid receptors as agonists and/or antagonists. We decided to explore the benzofuro[2,3-c]pyridin-6-ols3 because they appeared to be fairly rigid, partial structures of the oxide-bridged phenylmorphans. We synthesized the enantiomers ((þ)-15 and (-)-22, Scheme 3) of the most potent racemic compound (rac-cis-2-phenethyl-4a-ethyl-1,2,3,4,4a, 9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol) on a larger scale than previously reported for the racemate and determined which of the enantiomers was responsible for the high opioid receptor affinity. New N-substituted compounds with chiral side chains (16, 23, 17, and 24) were also prepared and evaluated to see if chiral N-hydroxyphenethyl moieties affect binding to opioid receptors. We then examined whether the active enantiomer topologically resembled any of our rigid oxide-bridged phenylmorphans utilizing quantum chemistry and a superposition study. Chemistry
† Dedicated to the 100th anniversary of the Division of Medicinal Chemistry. *To whom correspondence should be addressed. Phone: 301-4961856. Fax: 301-402-0589. E-mail:
[email protected]. a Abbreviations: para-d-oxide-bridged phenylmorphan, (3R*,6aS*11aR*)1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-8-ol; EI, electron impact; HRMS, high-resolution mass spectra; TLC, thin layer chromatography.
Synthesis and Resolution. The starting material, 2-hydroxy-5-methoxypropiophenone (1, Scheme 1), was isolated as a byproduct by Shulgin and Dyer,15 and their procedure for the preparation of 1 was used by Hutchison et al.3 We modified the procedure15 to obtain 1 in high yield on a large scale from the reaction between propionyl chloride and 1,4dimethoxybenzene, by using aluminum chloride with nitromethane as the solvent. Selective O-demethylation of the intermediate product, 2,5-dimethoxypropriophenone, was required in this reaction to obtain the desired monomethoxy compound 1, and preliminary experiments with BBr3 gave
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Figure 1. Twelve racemic N-methyl substituted oxide-bridged phenylmorphan isomers.
Scheme 1a
a Reagents and conditions: (a) propionyl chloride, AlCl3, CH3NO2, 60 C; (b) BrCH2CO2Et, K2CO3, acetone, reflux, 30 h; (c) (EtO)2POCH2CN, NaH, THF, 0 C, 2 h; (d) NaH, EtOH, reflux, 15 min; (e) H2, 5% Pt/C, acetic acid, 50 psi, room temperature; (f) 60% NaH in mineral oil, EtOH, reflux, 8.5 h; (g) NaBH4, (CH3O)2SO2, THF, reflux 48 h, purified through oxalate salt (oxalic acid, acetone), followed by KOH (aqueous) and distillation of the free base at 183 C, in vacuo (68%). The asterisk (/) indicates that the yield of (()-7 after the combination steps of e and f was about 60%.
unsatisfactory results. The use of anhydrous aluminum chloride in dry nitromethane at 60 C gave, in a rapid reaction, the desired product 1 in 84% yield, after workup. At a lower temperature (10 C) the reaction resulted in the formation of only the 2,5-dimethoxypropriophenone intermediate and little of the desired O-demethylated product 1. The reaction sequence to the known racemic secondary amine 8 (Scheme 1) was similar to that used by Hutchison
et al.3 but modified for the larger scale synthesis. The yield in the conversion of 1 to the propiophenone (2) was improved from 65%3 to 90% by running the reaction in acetone, monitoring its completion by TLC and NMR, and by crystallization using hexane and isopropanol. Borane reduction16 of the amide 7 gave the racemic cis-6-methoxyamine 8. Compound 8 was treated with (S)-(þ)-mandelic acid to give the salt with the (þ)-amine base 9 (Scheme 2). From the
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Figure 2. X-ray crystallographic structure of the R-(-)-mandelic acid salt of 4aS,9aR-(-)-cis-4a-ethyl-6-methoxy-1,2,3,4,4a,9ahexahydrobenzofuro[2,3-c]pyridine ((-)-10). Displacement ellipsoids are shown at the 50% level.
Scheme 2a
Scheme 3a
a
Reagents and conditions: (a) (S)-(þ)mandelic acid, EtOAc/Et2O (recrystallized from acetonitrile/Et2O (1:1) (1), EtOAc/Et2O (1:1) (1), and acetonitrile/Et2O (1:1) (1)), free-based with NaOH (2 N), extracted by CHCl3, solvent removed, high vacuum distilled at 130 C; (b) (R)-(-)mandelic acid, EtOAc/Et2O (recrystallized from EtOAc/ Et2O (1:1) (1), acetonitrile/Et2O (1:1) (2)), 68% recovery. Free-based with NaOH (2 N), extracted with CHCl3, solvent removed, high vacuum distilled at 125 C.
mother liquors of the recrystallization of the salt, material enriched in the (-)-enantiomer 10 was obtained and converted to the base, and treatment with (R)-(-)-mandelic acid gave the desired salt with the (-)-amine base 10 (Scheme 2). The enantiomers (þ)-9 and (-)-10 were obtained from their mandelate salts on basification. The melting points of these enantiomers were similar, and their optical rotations were about equal and opposite. Derivatization of (þ)-9 or (-)-10 with R-(þ)-1-phenethyl isocyanate to form the diastereomeric ureas17 enabled the determination of the optical purity of these amines. The optical purity of 9 was established by the disappearance of the chemical shift at δ 1.453 and that of 10 by the disappearance of the chemical shift at δ 1.408. Their optical purity was estimated to be ∼99% ee.17 The chirality of (-)-10 was determined to be 4aS,9aR by X-ray crystallographic analysis of the salt (-)-10 3 R-(-)mandelate (Figure 2). N-Alkylation of the secondary amine 4aR,9aS-9 or 4aS,9aR-10 (Scheme 3) gave the dextrorotary N-methyl (4aR,9aS-11) and N-phenylethyl analogue (4aR,9aS-12) and their levorotatory relatives (4aS,9aR-18 and 4aS,9aR-19). Cleavage of the aromatic methoxy group with 48% HBr gave the phenols, 4aR,9aS-14 and 4aR,9aS15 and 4aS,9aR-21 and 4aS,9aR-22 (Scheme 3). The (þ)-Sand R-20 -hydroxyphenethyl compounds 4aR,9aS-16 and 4aR,9aS-17 and the (-)-S- and R-20 -hydroxyphenethyl compounds 4aS,9aR-23 and 4aS,9aR-24 (Scheme 3) were
a Reagents and conditions: (a) 10% Pd/C, H2, 37% formic acid, MeOH, room temperature, 4 h; (b) (2-bromoethyl)benzene, K2CO3, DMF, 50 C, 20 h; (c) 48% HBr, reflux, 1 h, then NH4OH, CH2Cl2; (d) 48% HBr, reflux, 30 min, then NH4OH, CH2Cl2; (e) 48% HBr, HOAc, reflux, 7.5 h; (f) (S)-(-)-styrene oxide, toluene, reflux, 72 h; (g) (R)-(þ)styrene oxide, toluene, reflux, 72 h.
obtained from the 4aR,9aS-aminophenol 13 and the 4aS,9aR-aminophenol 20, respectively. These phenols (4aR,9aS-13 and 4aS,9aR-20) were prepared by 48% HBr cleavage of the aromatic methoxy secondary amine compounds 4aR,9aS-9 and 4aS,9aR-10. The various chiral compounds were generally prepared in moderate to high yields. Results and Discussion Five compounds (20-24) were examined for their efficacy as μ-agonists in the [35S]GTP-γ-S functional assay (Table 1). In that assay, the secondary amine 20 was found to be a weak partial agonist (ED50 = 150 nM) and had weak antagonist activity (Ke = 95 nM), the N-methyl substituted pyridin-6-ol 21 had good affinity for the μ-receptor (Ki = 16 nM) but had little efficacy in the functional assay, 23 was noted to be a weak partial agonist, compound 24 was a weak agonist, and the 4aS,9aR-N-phenethyl substituted pyridin-6-ol (22) was, as expected, a potent agonist with very high affinity for the μreceptor (Ki = 0.7 nM). As shown in Table 1, the 4aS,9aR compounds (all levorotatory) were found to have much higher affinity than the comparable 4aR,9aS compounds (all dextrorotatory). Clearly, the activity of the racemic mixtures
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Table 1. [3H]Opioid Receptor Binding Dataa for cis-Benzofuro[2,3-c]pyridin-6-ols and Functional Data ([35S]GTP-γ-S)b for Selected Compounds Ki (nM) μ
δd
κe
μ-agonism Emax (%)f
4aS,9aR 20g 21 22 23 (20 S) 24 (20 R) 4aR,9aS
48 ( 2.9 15.9 ( 1 0.70 ( 0.06 9.3 ( 0. 7 80 ( 5
1350 ( 90 75 ( 8.8 443 ( 32 807 ( 45
450 ( 34 88 ( 8 413 ( 44 1090 ( 60
97 ( 4 45 ( 2 84 ( 4 100 ( 3 36 ( 2
13 14 15 16 (20 S) 17 (20 R)
3500 ( 417 1330 ( 50 200 ( 9 600 ( 34 1330 ( 80
4270 ( 240 2690 ( 210 2490 ( 230 3100 ( 90
>10000 2325 ( 160 5870 ( 470 9500 ( 780
c
ED50 (nM) 150 ( 25 241 ( 47 24 ( 6 560 ( 60 480 ( 90
a Assays22 were conducted using CHO cells, which were stably transfected and express the μ-, δ-, or κ-opiate receptors, respectively. For all results, n = 3. For comparison, the Ki of morphine at μ is 2.55 ( 0.01 nM. b [35S]GTP-γ-S binding was performed using CHO hMOR cells, which express the human μ-opiate receptor. For all values, n = 3. c [3H]DAMGO binding. d [3H]DADLE binding. e [3H]U69,593 binding. f The Emax is the extrapolated maximal stimulation where 100% is defined as the stimulation produced by 10 μM DAMGO (DAMGO ED50 at μ is 42 ( 4 nM). g Secondary amine 20 is a μantagonist, Ke = 95 ( 16 nM ([35S]GTP-γ-S assay with CHO hMOR cells, n = 5).
examined by Hutchison et al.3 reflected the activity of the compounds with the 4aS,9aR stereochemistry. The 4aS,9aRN-phenethyl compound 22 had subnanomolar affinity for the μ-opioid receptor and slightly more than 100-fold less affinity at the δ- and κ-receptors. Interestingly, introduction of a hydroxy substituent in the N-phenethyl side chain decreased affinity for the μ-receptor 10-fold with the hydroxy in the Sconfiguration (4aS,9aR,2’S-23) and over 100-fold when the hydroxy was in the R-configuration (4aS,9aR,20 R-24). Figure 3 illustrates the geometry optimized conformers of the high affinity N-phenethyl compound (22) in the gaseous phase in its protonated form; the active form of 22 is likely to be protonated at physiological pH. For the comparison of the energetics, the Gibbs free energy of each conformer was calculated at 298.15 K from its respective fully optimized geometry in CHCl3. The topology of A, except for the equatorial N-phenylethyl substituent, is essentially identical to the X-ray structure of 10 as evidenced by the less than 0.04 A˚ root-mean-square deviation (rmsd) between the heavy atoms of both the dihydrofuran and the piperidine rings. Conformer B1 is epimeric to A and was obtained by nitrogen inversion; it is 2.6 kcal/mol less stable relative to A, a consequence of the change in the orientation of the Nphenylethyl from equatorial to axial. This nitrogen inversion in solution is likely to proceed through deprotonation followed by reprotonation. B2 was obtained by ∼180 rotation of B1 to point both the hydrogen on the nitrogen atom and the dihydrofuran ring of B1 out of the plane. A conformational change of the piperidine ring in B2 or B1 of Figure 3 via the variation of the dihedral angle of O9-C9a-C1-N2 (φ1) and C4a-C4-C3-N2 (φ2) leads to conformer C with the equatorially oriented N-phenylethyl moiety; conformer C is slightly (0.26 kcal/mol) more stable than A, and this stability may arise from the charge interaction between the dihydrofuran oxygen and the hydrogen on the nitrogen atom, as evidenced by their distance of 2.4 A˚. Note that A can undergo the piperidine conformation change first, followed by the nitrogen inversion to conformer C. Both the reaction pathway and the energy barrier between the two conformers A and C are not known and are currently being investigated with the ab initio replica path method.18 Nonetheless, considering that both the nitrogen inversion energy barrier and the conformation energy barrier of the neutral N-methylpiperidine are
known to be less than 11 kcal/mol,19,20 22 may well undergo a rapid interconversion between A and C and thus is likely to exist as a conformer mixture at room temperature. Energetics suggest that 22 exists in CHCl3 predominantly in the form of A and C (>95%) at room temperature. To identify the conformer most likely to be recognized at the μopioid receptor, the heavy atoms of the piperidine rings of A and C and the oxide-bridged para-d phenylmorphan isomer (which was found to be structurally more similar to 22 than any of the other oxide-bridged phenylmorphans) were fitted to morphine with the equatorial N-methyl as shown in Figure 4. Their respective rmsd value of the fitting is 0.13, 0.12, and 0.07 A˚. The dihydrofuran ring of conformer C overlaps well with that of the para-d isomer that is known to bind poorly to the μ-opioid receptor (Ki = 1220 nM).13 This indicates that conformer C, although thermochemically more stable than A, is not recognized by the receptor, and thus, the subnanomolar affinity of 22 (at μ, Ki = 0.7 nM) is likely to arise from conformer A. The benzofuran ring in conformer A overlaps better with the benzofuran ring of morphine than that of the para-d isomer (Figure 4). The affinity of 22 for the μ-receptor is much closer to that of morphine (Ki = 2.55 ( 0.1 nM) than to the para-d isomer. This work has enabled us to determine the enantiomer responsible for the μ-affinity and activity of the racemic Nsubstituted benzofuro[2,3-c]pyridin-6-ols.3 In addition, we have compared the structures of the energy-minimized conformers of the most active enantiomer 22 and the various oxide-bridged phenylmorphans, determining that the activity and affinity of 22 were not likely to be due to conformer C, which is structurally similar to the para-d-oxide-bridged phenylmorphan. Compound 22, however, was found to have a less stable conformer A, topologically similar to morphine, that could be responsible for its activity. Also, we synthesized new N-derivatives with a hydroxyl substituent on the phenylethyl side chain (23 and 24) and determined that a hydroxyl moiety on that side chain hindered binding and reduced efficacy. Experimental Section All reactions were performed in oven-dried glassware under an argon atmosphere. Some of the large scale mixtures were magnetically stirred. All melting points were determined on a
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Figure 3. Geometry optimized conformers of the protonated 4aS,9aR-(-)-cis-4a-ethyl-2-(2-phenylethyl)-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol (22) in the gaseous phase. Topology of A is essentially identical with the X-ray structure of 10 in Figure 2 (without the Nphenylethyl moiety). Conformer B1 is epimeric to A with respect to the nitrogen. B2 is another representation of B1 via a 180 rotation, and C is formed by a conformational change of the piperidine ring of B2 or B1. φ1 is the dihedral angle of O9-C9a-C1-N2, and φ2 is the dihedral angle of C4a-C4-C3-N2.
Thomas-Hoover capillary melting point apparatus and are uncorrected. The optical rotation data were obtained on a PerkinElmer polarimeter model 341 or on a Jasco DIP-370 digital polarimeter (589 nm). 1H NMR (in CDCl3 with TMS at δ 0.0 ppm) spectra were recorded on a Varian XL-300 spectrometer at 300 MHz and on a Varian AS-400 spectrometer at 400 MHz. Mass spectra (EI) and high-resolution mass spectra (HRMS) were obtained using a JEOL SX102 instrument. Thin layer chromatography (TLC) was performed on precoated plates of silica gel GF (0.25 mm, F254, Alltech) using various gradients of CHCl3/MeOH containing 1% NH4OH or gradients of ethyl acetate/n-hexanes. Visualization was accomplished under UV or by staining in an iodine chamber. Flash chromatography was conducted using silica gel (230-400 mesh, Merck). Elemental analyses were performed by Atlantic Microlabs Inc., Norcross, GA, to determine the purity of the compounds and were within (0.4% of theoretical values, confirming g95% purity. 2-Hydroxy-5-methoxypropiophenone (1). In a 5 L threenecked flask fitted with a mechanical stirrer, thermometer, nitrogen purge and pressure equalizing funnel was placed 500 mL of dry nitromethane. The stirred solvent was cooled to -20 C, and 277.82 g (2.08 mol, 1.15 equiv) of anhydrous AlCl3
was added slowly (exothermic) keeping the temperature at
